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G3326-01 / 05 / 15 2×Universal Blue SYBR Green qPCR Master Mix With 40×Yellow Template Dilution Buffer

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G3326-01 / 05 / 15 2×Universal Blue SYBR Green qPCR Master Mix With 40×Yellow Template Dilution Buffer

This product is a 2x premix solution for qPCR using SYBR Green I chimeric fluorescence method. The core component, Taq DNA Polymerase, is a heat-activated DNA Polymerase blocked by antibody method, which can effectively inhibit nonspecific amplification under low temperature conditions. Meanwhile, combined with the reaction Buffer optimized for qPCR, Taq DNA Polymerase is very suitable for high specificity and sensitivity qPCR reaction.

A good standard curve can be obtained in a wide quantitative area, which is accurate, reproducible and reliable for the quantitative analysis of target genes. At the same time, this product contains a special ROX Passive Reference Dye which is suitable for use in all qPCR instruments, and there is no need to adjust the ROX concentration on different instruments. Blue dye is added in the premix, which has the tracer effect of adding samples, and yellow template diluent is provided at the same time. When the template is diluted with yellow template diluent and added into the blue reaction premix, the color changes from blue to green, which can play a tracer role in the preparation of reaction system process and prevent leakage or misaddition. The spectra of the blue and yellow dyes do not overlap with the qPCR dyes and do not affect the reaction results.

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Product Variations:

Product code Description Qty
G3326-01 2xUniversal Blue SYBR Green qPCR Master Mix with 40xYellow Template Dilution Buffer 1mL
G3326-05 5x1mL
G3326-15 15x1mL

 

Package Contents:

Item code Description G3326-01 G3326-05 G3326-15
G3326-1 2x Universal Blue SYBR Green qPCR Master Mix 1mL / 5x1mL / 15x1mL 5x1mL 5x1mL
G3326-2 40x Yellow Template Dilution Buffer 1mL 1mL 1mL
  Manual 1pc 1pc 1pc

 

Technical Specification

Storage and Handling Conditions:

Wet ice bag transportation; Stored at -20ºC temperature, valid for 12 months.

Assay Protocol / Procedures:

1. (Optional) dilution template

This kit provides a 40x YELLOW Template Dilution Buffer, a 40-fold diluted Yellow Template Dilution that can be used to accurately determine whether the Template has been added to the qPCR reaction fluid by changing the color of the liquid. Taking the 20ul qPCR reaction system as an example, according to the dilution template amount added into the 20ul qPCR reaction solution, the corresponding dilution method of the original template can be referred to the following table (taking the original template diluted to 100ul as an example):

Add the diluted template to the 20ul qPCR reaction solution(ul)

1

2

3

4

5

6

7

8

Add the 40x YELLOW Template Dilution Buffer to the 100ul Template Dilution System(ul)

50

25

16.7

12.5

10

8.4

7.2

6.3

Add the 100ul Template Dilution System to the primary template(ul)

x

x

x

x

x

x

x

x

volume of adding Nuclease - Free Water(ul)

50-x

75-x

83.3-x

87.5-x

90-x

91.6-x

92.8-x

93.7-x

Example: 2ul diluted template should be added to 20ul qPCR reaction system. Taking the 100ul Template Dilution system as an example, when the original Template volume is 20ul, 25ul 40x Yellow Template Dilution Buffer is added, and then Nuclease-free Water 55ul is added to the total volume of 100ul. The 40x YELLOW TEMPLATE DILUTION BUFFER is now 10x. Add 2ul of the diluted Template into a 20ul Dilution Dilution Buffer, and the final 40× YELLOW Template Dilution Buffer is 1x. In conclusion, the Yellow Template Dilution Buffer should be 1x in the final qPCR system.

Note: If the template does not need to be diluted, or if the template diluent of G3362-2 is not used, you can ignore this step.

2. Recommend the qPCR reaction system:

Component 20µl rxn 50µl rxn Final Concentration

2×Universal Blue SYBR Green qPCR Master Mix

10µl

25µl

1×

Forward Primer (10uM)a

0.4µl

1µl

0.2µM

Reverse Primer (10uM)a

0.4µl

1µl

0.2µM

Templateb

Variable

Variable

as required

Nuclease-Free Water

Add to 20µl

Add to 50µl

  

a. Usually, a good amplification effect can be obtained with the final concentration of 0.2um. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1.0um.

b. The amount of template addition varies with the copy number of the target gene in the template solution, and the appropriate amount of template addition is discussed by gradient dilution. The best addition amount of template DNA in the 20ul reaction system was less than 100 ng. When the cDNA (RT reaction solution) of RT-PCR reaction was used as template, the addition amount should not exceed 10% of the total volume of PCR reaction solution.

3. PCR reaction procedure (can be adjusted according to the instruments) :

A. Two steps method B. Three steps method

Stage

Step

Cycle number

Temperature

Time

Stage

Step

Cycle number

Temperature

Time

Stage 1

Predegeneration

1

95ºC

30 sec

Stage 1

Predegeneration

1

95ºC

30 sec

Stage 2

Degeneration

40

 

95ºC

 

15 sec

Stage 2

Degeneration

40

95ºC

15 sec

annealing-extension

60ºC

30 seca

annealing

55-65ºC

10 sec
     

extension

72ºC

30 seca

Stage 3

melting curve

1

Instrument default Settings

Stage 3

melting curve

1

Instrument default Settings

a: If amplification specificity needs to be improved, two-step procedure or annealing temperature can be used; To improve the amplification efficiency, a three-step procedure or extension time can be used.
 

Note:

1. Please wear disposable gloves during operation to avoid RNase contamination.  

2. The reverse transcription products can be stored at -20ºC for a short time. If long-term storage is needed, it is recommended to store them at -80ºC after packing to avoid repeated freeze-thaw cycles.  

3. If the template is of eukaryotic origin, it is recommended to select Oligo (dT)18 Primer and pair it with the 3' Poly A tail of eukaryotic mRNA to obtain the highest yield of full-length cDNA.  

4. For reverse transcription of prokaryotic RNA, Random Hexamer Primer or Gene Specific Primer should be used.  

5. Random Hexamer Primer has wide applicability and is suitable for mRNA, rRNA, tRNA, small RNA and lncRNA templates.  

6. If reverse transcription is followed by qPCR assay, Oligo (dT)18 Primer and Random Hexamer Primer can be mixed to make cDNA synthesis efficiency in all regions of mRNA the same, which helps to improve the authenticity and repeatability of quantitative results.  

7. If the subsequent qPCR primers are designed across exons, the genome removal step can be omitted.
 

Compatibility:

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900 HT Fast, StepOne™, StepOne Plus™, 7500/7500 Fast, ViiA 7™, QuantStudio™ series, PikoRealTM Cycler;

Stratagene: Mx3000P®, 3005P™, 4000™;

Bio-Rad: CFX96™, CFX384™, iCycler iQ™, iQ5™, MyiQ™, MiniOpticon™, Opticon®, Opticon 2, Chromo4™;

Eppendorf: Realplex 2s, Mastercycler® ep, realplex;

IIIumina: Eco QPCR;

Cepheid: SmartCycler®;

Qiagen Corbett: Rotor-Gene® series;

Roche: LightCycler™ series;

Takara: Thermal Cycler Dice series;

Analytikjena: qTOWER series;

qTOWER: LineGene series.

 

Primer design principles:

1. The length of amplification product is recommended to be between 80-300 bp;  

2. Primer length: 18-25 bp;  

3. The content of base G+C in primers should be between 40%-60%;  

4. The Tm value difference between forward primers and reverse primers is less than 2ºC, and the Tm value between 58-62ºC is the best;  

5. Randomness of base distribution;  

6. Primers had better not contain self-complementary sequences, otherwise they will form a secondary hairpin structure;  

7. There should be no more than 4 complementary or homologous bases between two primers, otherwise primer dimer will be formed, especially complementary overlap at the 3' end;  

8. The 3' terminal base of the primer is suggested to be G or C;  

9. No other non-specific products were found in NCBI comparison results.

 

Common problems and solutions:

Problem description Possible reasons Solutions

At the end of the reaction, no amplification curve appeared or CT value appeared too late

The template concentration is too low

Repeat the experiment to reduce the template dilution multiple, and start from the highest concentration when the sample concentration is unknown

Template degradation

The template was prepared again and the experiment was repeated

  

There are PCR inhibitors in the system

Generally, the template is carried in, the dilution ratio of the template is increased or the template with high purity is reprepared and repeated

  

Primers may degrade

Primers that have not been used for a long time should first be tested for integrity by PAGE electrophoresis to rule out the possibility of degradation

  

Low amplification efficiency

ncrease the primer concentration, try a three-step amplification procedure, or redesign the primer

  

The amplification product is too long

The amplification product length was controlled in the range of 80-300 bp

  

The blank control shows the signal

Reaction system pollution

Firstly, the blank control water should be replaced. If the same situation still occurs, the primers, aspirators and PCR tubes should be replaced or a new Master Mix should be started. The reaction system is prepared in a super clean table to reduce aerosol pollution

Non-specific amplification such as primer dimers appears

Generally, it is normal for the amplification products to appear in blank control after 35 cycles, which should be analyzed with the melting curve.

Redesign primer, adjust primer concentration or optimize PCR reaction procedure

  

The melting curve has multiple peaks

Primer design is poor

The new primer was re-designed according to the primer design principles

Primer concentration is too high

Reduce primer concentration appropriately

  

There is genomic contamination in cDNA template

The extracted RNA solution is digested using DNA enzymes, such as DSDNase, to remove genomic contamination, or to design transintron primers

  

Poor reproducibility of experiments

The error of adding sample is large

The use of accurate pipette, with high quality suction head accurate pipette;

High dilution template, adding large volume template to reduce sampling error;

The reaction volume of qPCR was enlarged

The template concentration is too low

Repeat the experiment to reduce the dilution times of the template

  

Temperature deviation at different locations of the qPCR instrument

Calibrate the qPCR instrument regularly

  

The amplification curve is not smooth

Fluorescence signal is too weak, produced after system correction

Ensure that the dyes premixed in the Master Mix are not degraded;

Replace fluorescent signal to collect better qPCR consumables

Amplification curve breaks or slips

The template concentration was higher and the baseline endpoint value was greater than the CT value

The baseline endpoint (Ct value -3) was reduced and the data were reanalyzed

Amplification curves of individual Wells suddenly dropped sharply

There are bubbles in the reaction tube

Ensure that MIX is completely dissolved, and do not swirl and oscillate evenly;

After the sample is added, the bubbles are removed by centrifugation with light elastic.

The pre-denaturation time was extended to 10 min to remove the bubbles
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